Root evaluation

ABSTRACT

A method is provided comprising (i) growing a plant in a substantially transparent container charged with a particulate, non-transparent growing medium; and (ii) evaluating plant roots through said transparent container by digital imaging. An apparatus for evaluating plant roots in a high throughput manner is also provided.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP2005/054466 filed Sep. 8, 2005, which claims the benefit ofEuropean application 04104496.7 filed Sep. 16, 2004.

The present invention relates generally to the field of plant breedingand concerns a method for evaluating plant roots by growing plants in asubstantially transparent container charged with a particulate,non-transparent growing medium and evaluating plant roots through thesubstantially transparent container by digital imaging. The inventionalso provides apparatus for evaluating plant roots, which apparatus isparticularly suitable for evaluating, in a high throughput fashion, theroots of plants growing in a greenhouse.

Plant breeding starts in essence with the creation of genotypicvariation among plants of a given species. The creation of genotypicvariation relies on the production of genetic alterations that can beobtained by various techniques including recombination through classicalcrossing, chemical mutagenesis, radiation-induced mutation, somatichybridisation, inter-specific crossing and genetic engineering.Following the creation of genotypic variation, selection of thosegenotypes having the most desirable agronomic phenotypes is performed.For example, plants may be selected for certain root phenotypes.

Despite the great importance of roots to the overall survivability of aplant, the evaluation of root systems, particularly that of mature rootsystems, is often hindered by technical limitations. In a field setting,a traditional method for evaluating root systems involves excavating theroots from the soil before performing any measurements; this means thatevaluation may take place only once for any given plant. Furthermore,variation over short distances in the soil (due to, for example, soilcomposition, physical properties of the soil, availability of nutrientsor the presence of microbial soil inhabitants) may also hinder rootevaluation in a field setting.

Several methods are available for the non-destructive measurement ofroots. One such method relies on the use of electrical capacitance whichinvolves inserting electrodes in the plant (preferably the stem) and thesubstrate. This allows measurement of the electrical properties of thecontact surface between the roots and the substrate, and from this theroot mass is deduced. There are however disadvantages to this technique.For example: i) reliability is low due to difficulties associated withcalibration (for example, varying water content of the substrate); ii)insertion of the electrode into the stem is difficult to automate,especially in plants with many stems (this applies to most cereals, forexample wheat and rice); iii) the only information generated using thistechnique is an indication of root mass.

Another method for measuring roots in a non-destructive manner involvesthe use of X-rays or nuclear magnetic resonance (NMR), however thesetechniques can be extremely slow and expensive and are thereforegenerally not suitable for use where a high throughput is required.

A further method for measuring roots in a non-destructive mannerinvolves the use of a ground-penetrating radar, however the lowresolution associated with use of this technique makes the resultsdifficult to interpret.

Although plant breeders are generally aware of the importance of rootsystems, the shortcomings associated with traditional methods forevaluating root systems means that plant breeders tend to focus theirattention on the aboveground parts of plants, with little if anyattention being paid to belowground parts. Much of the current knowledgeof root function has been gained from studies of seedlings or inferredfrom observations to the plant canopy and soil profile.

The ability to evaluate plant roots without the disadvantages of theprior art would enable plant breeders to gain knowledge of, for example,the profile of an optimal root system and how it should be proportionedwith respect to the rest of the plant. It would also be useful forbreeders to understand what the most efficient root architecture for agiven set of environmental conditions would be. For example, it iswidely believed that plants with a more developed root system shouldhave higher resistance to drought stress. However, many questionsremain, such as the degree of development required, whether there areany penalties to the rest of the plant as a consequence of having a moredeveloped root system and the optimum ratio of root to leaf area. Theability to evaluate plant roots without the disadvantages of the priorart would enable plant breeders to answer these and other suchquestions.

It is therefore an object of the present invention to provide means toevaluate plant roots which alleviate some of the aforementionedproblems.

Therefore, according to the present invention, there is provided amethod for evaluating plant roots, comprising (i) growing a plant in asubstantially transparent container charged with a particulate,non-transparent growing medium; and (ii) and evaluating the plant rootsthrough the substantially transparent container by digital imaging.

The substantially transparent container may be a pot, tray, or the like.Preferably, the container is a classical plant pot moulded oftransparent material, such as a suitable plastics material. It ispreferred to have one plant per pot. The transparent material preferablycontains a (green) pigment to absorb substantially all wavelengths oflight except those between 500 and 600 nanometres. This serves tosuppress the light avoidance response of the roots and to avoid algalgrowth on the inner walls of the pots. In use, plant roots may be seenagainst the walls of the container. The container may consistsubstantially entirely of transparent material or may have only atransparent bottom.

The particulate, non-transparent growing medium may be any soil-likesubstrate (for example soil, compost, peat, sand, or the like, or acombination thereof). The use of a defined substrate such as pottingsoil or rock wool may further reduce variations caused by theheterogeneity of the soil. Furthermore, the growing of greenhouse plantson a defined substrate allows the amount of water and nutrients to begiven to the plants to be controlled.

According to the method of the invention, plant roots are evaluated bydigital imaging, which facilitates the handling of large samples (of theorder of say up to several tens of thousands). It is preferred thatplants are evaluated in an automated fashion using at least one camera.The plants may be presented (sequentially) to fixed camera(s), or thecamera(s) may be moveable to plants in a fixed spot. According to apreferred aspect of the present invention, the plants are retrieved (forexample from a location in the greenhouse or elsewhere) and thesubstantially transparent containers in which the plants are placed arepresented sequentially to a camera arranged to record images of theroots. The plants are then returned to their original location, or toanother location, all substantially without human intervention. Theremay be some degree of human intervention in the step of selecting theplant or series of plants for evaluation, but this may also becomputerised. In a preferred process, a minimum of 500 plants per hourmay be evaluated.

According to a second aspect of the present invention there is providedan apparatus for evaluating plant roots, comprising:

-   -   (i) a transporter means for movement of plants in substantially        transparent containers charged with particulate, non-transparent        growing medium; and    -   (ii) at least one camera; and optionally    -   (iii) a mechanical system for presenting the substantially        transparent container to the at least one camera; and    -   (iv) algorithms for measuring root traits.

The transporter means is preferably a moving belt. An alternativeembodiment may also be envisaged in which the at least one camera ismoveable so as to record images of plants in a fixed spot. There istherefore provided apparatus for evaluating the roots of plants insubstantially transparent containers charged with particulate,non-transparent growing medium, which apparatus comprises at least onemovable camera arranged to record images of plant roots through saidsubstantially transparent container.

Automated evaluation may further be aided by having several plants, oreach plant, carry a unique identifier allowing information concerningthose individual plants to be linked to their unique identifier in acomputer. Preferably, the identifier is a transponder, but any othersuitable identifier such as a barcode may be used. Information may becollected and stored concerning the plant species, the cultivar,parental information, presence/absence of a transgene, sowing date, rootmeasurements (with a date stamp) and any other measurements orobservations made on parts of the plant other than the roots, also witha date stamp. The information stored in a computer may be retrieved byappropriate software. A statistical analysis may be made to identifyplants with differing root characteristics and to identify interestingtraits.

Examples of interesting traits include: (i) increased root growth rate(this is useful to know since cultivars having rapidly developing rootsystems are more efficient at taking up water, minerals and othernutrients during the critical early stages in the plant life cycle andin case of water shortage); (ii) increased root length and thickness(longer, thicker roots have a higher capacity to penetrate hardsubstrates and provide better adaptation to compacted soil, especiallyin case of water shortage); (iii) increased root branching (highlybranched root systems have a higher potential for efficient explorationof the soil layers); (iv) improved anchorage (root lodging reduces cropquality and yields; plants with sturdier root systems are less sensitiveto root lodging).

The method of the invention may be performed on any plant. Preferredplants are cereals, such as rice, wheat, and corn.

The method of the invention is particularly suited to the breeding ofplants in a greenhouse, the advantage being that plants are grown on adefined substrate, so that the amount of water and nutrients given tothe plants can be controlled. The method of the invention is alsoparticularly suited where a high throughput is required.

Preferably also, plants are selected for further breeding or forcommercial use by comparing the phenotype characteristics of the plants.

The word “comprising” where used herein is intended to encompass thenotion of “including” and the notion of “consisting essentially of”.

The present invention will now be described with reference to thefollowing drawings in which:

FIG. 1 is a diagrammatic representation (top view) of the apparatusaccording to a first embodiment of the invention.

FIG. 2 is a diagrammatic representation (side view) of the apparatusaccording to a first embodiment of the invention.

FIG. 3 is a diagrammatic representation (top view) of the apparatusaccording to a second embodiment of the invention.

FIG. 4 is a diagrammatic representation (elevated side view) of theapparatus according to a second embodiment of the invention.

FIG. 5 is a diagrammatic representation of the apparatus according to athird embodiment of the invention.

Each of FIGS. 1 to 4 is made up of three drawings in which the processof imaging plant roots is illustrated starting from the left handdrawing which shows the apparatus before imaging; the middle drawingwhich shows elements of the apparatus during imaging; and the drawing onthe right hand side which shows elements of the apparatus aftercompletion of imaging. The arrows indicate the direction of the movementof the mobile elements.

In order that the invention may become clearer there now follows adescription to illustrate the invention by way of example and to be readwith the accompanying drawings.

THE FIRST EMBODIMENT OF THE APPARATUS ACCORDING TO THE INVENTIONCOMPRISES

A first transporter means (1), by which the plants in transparent potsare supported and moved to the station where images of the roots arerecorded. Preferably, the transporter means is a moving belt. Containers(2) are plant pots in which one or more plants is growing in a mediumselected for the purpose. The pots are of a transparent plastic or thelike. A rotating plate (3) is shown, on which the potted plants can beplaced and rotated along a central vertical axis. A first mechanicaldevice, referred to as “pusher” (4) has the capability to move thepotted plant from the first transporter means (1) onto rotating plate(3). A camera (5) for recording pictures from the side surface of thepots is placed horizontally at a suitable distance from the rotatingplate (3). The camera is a “line-scan” type and can output images in adigital format. Two-dimensional digital pictures are constituted bystacked lines of pixels, the first dimension being the length of thelines and the second dimension the number of lines. One property ofline-scan cameras is that they record single lines of pixels at regulartime intervals and compile them during a specified period of time.According to this principle, it is possible to record an image of thesurface of a circular object by rotating the object in front of aline-scan camera. An appropriate light source (6) placed horizontally ata suitable distance from the rotating plate (3) so as to illuminate in auniform way the side surface of the pot.

A computer (7) is connected to the aforementioned camera and is equippedwith appropriate programs for collecting, storing, and analysing thepictures produced by the camera. Reading means (8) allow reading of theidentifier attached to the plant. Preferably, the identifier is atransponder, but any other suitable system can be used. A secondtransporter means (9) transports the plant pots to their originallocation or to another location if desired. This transporter means isalso a moving belt. A second mechanical device, referred to as “pusher”(10), has the capability to move the potted plants from the rotatingplate (3) onto the second transporter means (9).

The process by which the root traits are evaluated in the firstillustrative apparatus typically include the following steps:

The first transporter means (1) is set in motion in order to bring apotted plant (2) in front of the rotating plate (3). The firsttransporter means (1) is then stopped. The first pusher (4) is activatedand pushes the potted plant onto the rotating plate (3). Alternatively,the action of placing the plant on the rotating plate may be performedmanually or by a robotic arm with a suitable gripper to seize the plantand to move it to a pre-selected place. The potted plant is turnedaround in front of the line-scan camera (5) and the light-source (6) isswitched on. Preferably, the light-source (6) is not switched offbetween each plant to ensure a stable illumination at each cycle ofpicture acquisition. The line-scan camera (5) starts recording a pictureas soon as the rotating plate (3) is turning and stops after a completerevolution. The identifier attached to the potted plant is read by theidentifier reader (8) and sent through a data cable connection to thecomputer (7). The camera (5) sends the recorded picture through a datacable connection to the computer (7). The identifier of the plant islinked to the image at the moment the image is stored and analysed.Preferably, the identifier is incorporated in the name of the image filewhen the image file is stored, but any naming system can be used to namethe images, provided that the link between the identifier and the imagename is linked unequivocally in a computer database. The root featuresare deduced from the picture by means of appropriate software and theresult is stored in a computer database with a link to the identifier ofthe plant. The second pusher (10) pushes the potted plant onto thesecond transporter means (9) which is then set in motion to transportthe potted plant. Alternatively, the action of moving the plant backfrom the rotating plate to the second transporter means can be performedeither manually or by a robotic arm with a suitable gripper that canseize the plant and move it to pre-selected places. The above steps arerepeated until all the plants placed on the first transporter means havebeen evaluated.

THE SECOND ILLUSTRATIVE APPARATUS COMPRISES

A transporter means (21) on which plant pots are supported and moved toa station where images of the roots are recorded. Preferably, thetransporter means is a moving belt. Containers (22) in the form of plantpots in which one or more plants is growing in a medium selected for thepurpose. The material of the pots is preferably a transparent plastic orthe like. A mechanical device, referred to as “gripper-arm” (23) thathas the capability to seize the potted plants from the transporter means(21) and to move them over short distances, for example between 10 and50 cm, both horizontally in the direction perpendicular to thetransporter means (21) and vertically in the direction perpendicular tothe ground. A water-basin (24) with a transparent bottom is provided.The water-basin can be of any shape, but its length and width must be atleast greater than the diameter of the pots. Preferably, the height ofthe water basin should not exceed the height of the pots. A camera (25)placed below the water-basin (24) records pictures of the bottom surfaceof the pots. T Camera (25) outputs images in a digital format.Appropriate light sources (26) are placed at a suitable distance belowthe water-basin (24) so as to illuminate in a uniform way the bottomsurface of the pot. A computer (27) is connected to the aforementionedcamera, and equipped with the appropriate programs for collecting,storing, and analysing the pictures produced by the camera. Means toread the identifier attached to the plant (28) are provided. Preferably,this is a transponder, but any other suitable system may be used.

The process by which the root traits are evaluated in the secondillustrative apparatus typically include the following steps:

The water basin (24), camera (25), and gripper-arm (23) are installed ata suitable distance from the transporter means (21) on which the pottedplants (22) are placed for transportation. The transporter means (21) isset in motion to bring a plant in front of the water basin (24). Thetransporter means (21) is then stopped.

The gripper arm (23) is activated and seizes the plant pot placed infront of the water basin (24). The plant pot is moved by means of thegripper arm (23), first horizontally above the water basin (24), thenvertically downwards into the water basin (24). Plunging the pot intothe water basin removes dust, soil or condensed water from the bottomsurface of the pot so that image quality is improved. Preferably, acontinuous stream of water is maintained in the water basin toaccelerate the cleaning process. Alternatively, the action of placingthe plant in the basin may be performed manually. Alternatively, thewater basin may be a part of the transporter means and the pot may beguided over the basin by means of a robotic arm and lateral rails. Theidentifier attached to the potted plant is read by the identifier reader(28) and sent through a cable connection to a computer (27). The camera(25) located under the water basin records an image of the bottom of thepot. The image is sent through a cable connection to a computer (27).The identifier of the plant is linked to the image at the moment theimage is stored and analysed. Preferably, the identifier is written inthe name of the image file when the image file is stored, but any namingsystem can be used to name the images, provided that the link betweenthe identifier and the image name is linked in a computer database. Theroot features are deduced from the picture by means of appropriatesoftware and the result is stored in a computer database with a link tothe identifier of the plant. The plant pot is placed back on thetransporter means (21) which is set in motion again in order to bringthe next plant in front of the water basin (24). Alternatively, theaction of placing the plant back on the transporter means can beperformed manually. The aforementioned Steps are repeated until all theplants placed on the transporter means (21) have been evaluated.

THE THIRD ILLUSTRATIVE APPARATUS COMPRISES

A holder, or tray, (31), by which the plant pots are supported and movedto the station where images of the roots are recorded. Preferably, theholder dimensions should comply with industrial standards such as thepopular “Danish trays”. Typically, the holder would be approx. 50 cmlong by 30 cm wide and 5 to 10 cm high. The transport and handling ofthe trays may be performed using transporter means and robotizedmechanical devices that are commercially available for the horticulturalindustry. In the present example, such a tray consists of a plasticframe with 8 circles where pots of 12 cm diameter can be inserted. Thesystem can be scaled up or down for use with different species ofplants. Containers (32) in the form of plant pots in which one or moreplants is growing in a medium selected for the purpose. The pots are ofa transparent plastic material or the like. A digital imaging stationcomprising an appropriate camera (33) for recording pictures of thebottom surface of all the pots in the holder, placed vertically at asuitable distance from the plane where the pots will be positioned.Preferably, the camera can output images in a digital format.Appropriate light sources (34) placed at a suitable distance below theplane where the pots will be positioned so as to illuminate in a uniformway the bottom surface of the pots. A computer (35), connected to theaforementioned camera, and equipped with the appropriate programs forcollecting, storing, and analysing the pictures produced by the camera.In this example, each picture needs to be divided in sectorscorresponding to each individual plant. In this example, theidentification of the individual plants may be carried out using areadable tag, preferably in the form of a “bar code” or even morepreferably in the form of a “matrix code” under the bottom of eachindividual pot since transponders may not be suitable for use withplants in such close proximity to one another. Alternatively, thereadable tag may used to identify the tray; in this case, the individualplants on the tray could be identified by their positions on the tray.Most preferably, the tag and the glue, or any other mean used to attachthe tag to the bottom of the pot or tray, should be resistant to waterand to mechanical stresses.

The process by which the root traits are evaluated in the thirdillustrative apparatus typically include the following steps:

The holders (31) supporting the transparent pots (32) are brought to theimaging station and placed manually, or using mechanical transportsystems available in the horticultural industry, above the camera (33),at a suitable distance from the focal plane of the camera. The camera(33) located under the holder records a picture of the bottoms of thepots. If a higher resolution is needed, the camera can record the imageof one individual pot at a time, which requires sequential movements ofthe tray or the camera so that for each successive pot the vertical axisof the pot is made perpendicular to the focal plane of the camera.Several cameras may be used, for example one for each pot. The image issent through a cable connection to a computer (35). The identifier ofthe plant, which is present on the picture is recognized by means ofsoftware and is linked to the image at the moment the image is storedand analysed. Preferably, the identifier is written in the name of theimage file when the image file is stored, but any naming system can beused to name the images, provided that the link between the identifierand the image name is linked in a computer database. The root featuresare deduced from the picture, by means of appropriate software, and theresult is stored in a computer database with a link to the identifier ofthe plant. The holder containing the plants is returned to its originallocation, either manually or using mechanical transport systemsavailable in the horticultural industry.

1. A method for non-destructive evaluation of roots of potted plants toidentify plants possessing a trait of interest, comprising: (i) growingone or more plants in a container in which at least the bottom issubstantially transparent, and wherein the container is charged with aparticulate, non-transparent growing medium; (ii) sequentiallypositioning the container with one or more plants for digital imaging ofthe roots of the plants by a camera below the bottom of the container;and (iii) storing and analyzing information obtained from the plants toidentify plants possessing a trait of interest.
 2. The method of claim1, wherein the entire container is substantially transparent.
 3. Themethod of claim 1, wherein at least the bottom of said containercontains a pigment that absorbs all light wavelengths except between 500and 600 nanometers.
 4. The method of claim 1, wherein said particulate,non-transparent growing medium comprises soil.
 5. The method of claim 1,wherein the evaluation of plant roots is accomplished in a highthroughput manner.
 6. The method of claim 1, wherein each plant isprovided with an unique identifier.
 7. The method of claim 6, whereininformation concerning the plant is associated with the uniqueidentifier.
 8. The method of claim 7, wherein the information is storedin a computer.
 9. The method of claim 6, wherein said unique identifieris a transponder.
 10. The method of claim 1, wherein the method furthercomprises using a statistical analysis to identify plants with differingroot characteristics and identifying a plant with a trait of interest.11. The method of claim 10, wherein the method further comprisesselecting the plant with a trait of interest for further breeding or forcommercial use.
 12. The method of claim 10, wherein the statisticalanalysis is conducted with the aid of a computer.
 13. The method ofclaim 10, wherein the trait of interest is (i) increased root growth,(ii) increased root growth rate, (iii) increased root length, (iv)increased root thickness, (v) increased root branching, and/or (vi)improved anchorage.
 14. The method of claim 1, wherein the evaluation ofplant roots comprises evaluating one or more root characteristicsselected from (i) root growth, (ii) root growth rate, (iii) root length,(iv) root thickness, (v) root branching, and/or (vi) anchorage.
 15. Themethod of claim 1, wherein the camera is a line-scan camera that canoutput images in a digital format.
 16. A method for breeding plants in agreenhouse comprising: (i) growing plants and evaluating roots of theplants according to the method of claim 1; (ii) identifying plants withdifferent root traits, and (iii) selecting plants having desirable roottraits by comparing the root traits of the plants, and (iv) furtherbreeding the plants selected.
 17. An apparatus for evaluating plantroots, comprising: (i) one or more containers in which at least thebottom is substantially transparent, wherein the one or more containersare charged with particulate, non-transparent growing medium for growingplants therein; (ii) a camera that can output images in a digitalformat; and (iii) a transporter for movement of the one or morecontainers or the camera; wherein the transporter positions the one ormore containers or the camera for digital imagining by said camera belowthe bottom of the one or more containers.
 18. The apparatus of claim 17,wherein one or more plants are grown in each of the one or morecontainers, and wherein said one or more plants are associated with aunique identifier.
 19. The apparatus of claim 18, wherein said uniqueidentifier is a transponder.
 20. The apparatus of claim 17, wherein saidtransporter comprises a moving belt.
 21. The apparatus of claim 17,wherein the camera is a line-scan camera.
 22. A high-throughput methodfor non-destructive evaluation of roots of potted plants to identifyplants possessing a trait of interest, comprising: (i) growing plants ina substantially transparent container charged with a particulate,non-transparent growing medium; (ii) providing each plant with a uniqueidentifier; (iii) sequentially positioning each plant for digitalimaging by a camera positioned below the bottom of the container of theroots through the transparent container and obtaining informationconcerning the roots of each plant associated with the plant's uniqueidentifier; (iv) providing the obtained information for each plant to anassociated computer for identifying plants possessing a trait ofinterest.
 23. A method for non-destructive evaluation of roots of pottedplants in a high throughput manner to identify plants possessing a traitof interest, comprising: (i) growing one or more plants in a containerin which at least the bottom is substantially transparent and contains apigment that absorbs all light wavelengths except between 500 and 600nanometres, wherein the container is charged with a particulate,non-transparent growing medium comprising soil, and wherein each plantis provided with an unique identifier; (ii) sequentially positioning thecontainer with one or more plants for digital imaging of the roots ofthe plants by a camera below the bottom of the container, wherein thecamera is a line-scan camera that can output images in a digital format;and (iii) storing and analyzing information obtained from the plants toidentify plants possessing a trait of interest.